Interactions between the T cell surface molecule CD28 and its ligand B7 on the antigen presenting cell provide costimulatory signals that are important in determining the response of a T cell to antigen specific signals: only in the presence of the costimulatory signals are T cells fully activated to produce IL2 and divide. For many T cell clones, the lack of the CD28 signal can result in the induction of clonal anergy. This has led to the notion that this two signal requirement provides a mechanism for peripheral tolerance. Despite the considerable advances in the past three years, there are several issues which are as yet unanswered,, The role of anergy in peripheral tolerance remains to be established, and it is not yet clear that naive T cells can be anergized. Recent evidence suggests the existence of additional, as yet unidentified costimulatory ligands. Finally, the cD28 signaling pathway remains obscure. We propose: 1. To determine whether naive T cells are susceptible to induction of clonal anergy upon an initial encounter with antigen in the absence of costimulatory signals, or whether full activation is necessary to drive the differentiation of cells into an anergizable state. This will be accomplished by assessing the functional and phenotypic consequences of the sequential exposure of freshly isolated T cells from TCR transgenic mice to carefully regulated stimuli in vitro. 2. To functionally and phenotypically characterize the T cell response to immunization with soluble peptide in normal mice in order to gain insight into mechanisms of induction of tolerance, and to evaluate the consequences of manipulation of costimulatory signals in the response of TCR transgenic mice to peptide antigens. 3. To construct B7 transgenic mice in order to determine whether expression of B7 on inappropriate tissues results in autoimmunity. 4. To use an antibody that we have produced to the murine B7 gene product in combination with CTLA4-Ig to determine whether there are additional costimulatory ligands. If this proves to be the case, as is expected, then we will attempt the molecular cloning of the additional ligands and prepare monoclonal antibodies for use in characterization of the distribution and function. 5. To determine whether costimulatory signals are required for induction of cytotoxic activity in pre-CTL from T cell transgenic mice. 6. To gain insight into CD28 signaling by: (a) Characterizing the substrates of protein tyrosine kinase (PTK) phosphorylated in response to C28 signaling in freshly isolated and anergized T cells from TCR transgenic mice, and (b) using site directed mutagenesis to determine the role of the cytoplasmic domain of CD28 in signal transduction.